Campylobacter immunogenic compositions and uses thereof

ABSTRACT

The present disclosure provides immunogenic compositions against  Campylobacter  and methods for using the immunogenic composition to generate an immune response against  Campylobacter  and/or reduce intestinal colonization by  Campylobacter.

CROSS REFERENCE

This application is a divisional of U.S. application Ser. No. 14/375,365filed Jul. 29, 2014, which is a US National Phase filing ofPCT/US13/24332 filed Feb. 1, 2013, which claims priority to U.S.Provisional Patent Application Ser. Nos. 61/632,888 filed Feb. 1, 2012and 61/689,078 filed May 29, 2012, all incorporated by reference hereinin their entirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under grant number127032 awarded by United States Department of Agriculture Hatch Funding.The government has certain rights in the invention.

BACKGROUND

Campylobacteriosis is primarily a food-borne disease with the handlingand consumption of poultry considered to be the most significant riskfactor in transmission. Infection by Campylobacter spp. is one of theleading causes of bacterial gastroenteritis, causing an estimated 1.3million cases annually in the U.S. (Scallan et al, 2011), resulting inhealth care costs of $0.8-5.6 billion per year (Buzby et al., 1997).Serious complications such as arthritis occur in an estimated 1-5% ofcases (Pope et al., 2007)) and Guillain-Barre Syndrome, a form ofneuromuscular paralysis, occurs at a rate of 1.0 per 1,000 patients(Altekruse and Tollefson, 2003). Due to the emergence and persistence ofantibiotic resistance coupled with increasing regulatory restrictions onthe industry, control strategies such as vaccination are urgentlyneeded. To date, there is no intervention method or vaccine available tothe producer to reduce numbers of Campylobacter in poultry going toprocessing.

Beginning Jul. 1, 2011, USDA-FSIS (Food Safety and Inspection Service)has implemented new performance standards for Campylobacter for youngchicken chilled carcasses at slaughter establishments (FSIS NOTICE,31-11, Jun. 30, 2011). These standards will allow no more than 8positive Campylobacter samples out of a 51-sample set, with plans toinitially warn the companies and fines to be imposed in 2013. A largebaseline study was conducted in our laboratory (funded by the USDA) toquantify Campylobacter levels in slaughtering plants from 2007-2009. Ourstudies demonstrated 21.9% (213/972) of post-chilled carcass rinsesamples were positive for Campylobacter, which amounts to 11.17 per51-sample set. Clearly, this is over the allowable number ofCampylobacter from chilled carcass samples and will precipitate theissuing of fines for both the producer and processing plant unless areduction of Campylobacter in poultry can be obtained. Currently, thereare no available intervention methods or vaccines available forproducers to use to reduce the Campylobacter load in poultry.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides immunogeniccompositions, comprising one or more expression vectors comprising:

(a) at least one polynucleotide encoding a protein selected from thegroup consisting of proteins comprising an amino acid sequence at least80 percent identical to SEQ ID NO:2 (Cj0998c protein), SEQ ID NO:4(Cj0588 protein), and SEQ ID NO:6 (Cj0248 protein), or antigenicportions thereof; and

(b) a promoter operatively linked to the polynucleotide, wherein thepromoter region is capable of directing expression of the encodedprotein(s).

In a second aspect, the present invention provides immunogeniccompositions, comprising

(a) one or more isolated proteins selected from the group consisting ofproteins comprising an amino acid sequence at least 80 percent identicalto SEQ ID NO:2 (Cj0998c protein), SEQ ID NO:4 (Cj0588 protein), and SEQID NO:6 (Cj0248 protein), or antigenic portions thereof; and

(b) a pharmaceutically acceptable carrier.

In a third aspect, the present invention provides methods forstimulating an immune response against Campylobacter, comprisingadministering to a subject an effective amount of the immunogeniccomposition according to any embodiment of the invention to generate animmune response against Campylobacter.

In a fourth aspect, the present invention provides methods for reducingCampylobacter intestinal colonization in a subject, comprisingadministering an amount effective of the immunogenic compositionaccording to any embodiment of the invention to reduce Campylobacterintestinal colonization in the subject.

DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic drawing of the pYA3493 plasmid.

DETAILED DESCRIPTION OF THE INVENTION

All references cited are herein incorporated by reference in theirentirety. Within this application, unless otherwise stated, thetechniques utilized may be found in any of several well-known referencessuch as: Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989,Cold Spring Harbor Laboratory Press), Gene Expression Technology(Methods in Enzymology, Vol. 185, edited by D. Goeddel, 1991. AcademicPress, San Diego, Calif.), “Guide to Protein Purification” in Methods inEnzymology (M. P. Deutshcer, ed., (1990) Academic Press, Inc.); PCRProtocols: A Guide to Methods and Applications (Innis, et al. 1990.Academic Press, San Diego, Calif.), Culture of Animal Cells: A Manual ofBasic Technique, 2^(nd) Ed. (R. I. Freshney. 1987. Liss, Inc. New York,N.Y.), Gene Transfer and Expression Protocols, pp. 109-128, ed. E. J.Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.).

As used herein, the singular forms “a”, “an” and “the” include pluralreferents unless the context clearly dictates otherwise. “And” as usedherein is interchangeably used with “or” unless expressly statedotherwise.

All embodiments of any aspect of the invention can be used incombination, unless the context clearly dictates otherwise.

In a first aspect, the present invention provides immunogeniccompositions, comprising one or more expression vectors comprising:

(i) at least one polynucleotide encoding a protein selected from thegroup consisting of proteins comprising an amino acid sequence at least80 percent identical to SEQ ID NO:2 (Cj0998c protein), SEQ ID NO:4(Cj0588 protein), and SEQ ID NO:6 (Cj0248 protein), or antigenicportions thereof; and

(ii) a promoter operatively linked to the polynucleotide, wherein thepromoter region is capable of directing expression of the encodedprotein(s).

The inventors have identified three putative virulence genes (Cj0248,Cj0588, and Cj0998c) from C. jejuni encoding novel proteins from theouter-membrane of the bacterium. The inventors have further discoveredthat each of the Cj0998c protein, the Cj0588 protein, and the Cj0248protein are potent immunogens for stimulating an effective immuneresponse against Campylobacter jejuni (“C. jejuni”). For example, asdisclosed in detail herein, two separate vaccination trials of chickenswith a vector expressing the Cj0988c protein demonstrated reducednumbers of C. jejuni in birds after challenge an average of 2.5 logs CFU(geomean 3 logs) when compared to the cecal numbers of non-vaccinatedcontrol birds. Furthermore, vaccination trials demonstrated asignificant reduction (1-4 logs) (1 log with heterologous strain and 4logs with homologous strain) of C. jejuni in cecal contents of chickensvaccinated with vectors expressing the Cj0588 protein and challengedwith Campylobacter jejuni. Each of the proteins was initially isolatedfrom an outer-membrane (OMP) extraction of a C. jejuni biofilm.

Thus, the immunogenic compositions of the invention can be used, forexample for stimulating an immune response in subjects at risk ofCampylobacter infection and/or colonization, including but not limitedto vertebrates such as chickens, turkeys, birds, cattle, sheep, pigs,dogs, cats, and humans.

In one embodiment, the one or more expression vectors encode one of therecited proteins, or antigenic fragments thereof. In another embodiment,the one or more expression vectors encode two proteins comprising anamino acid sequence at least 80 percent identical to the recited aminoacid sequences (i.e.: SEQ ID NO:2 and SEQ ID NO:4; SEQ ID NO:2 and SEQID NO:6; or SEQ ID NO:6 and SEQ ID NO:8), or antigenic fragmentsthereof. In this embodiment, a single expression vector may encode bothproteins, or antigenic fragments thereof, or the composition maycomprise two expression vectors, with each expression vector encodingone of the recited proteins, or antigenic fragments thereof. In thisembodiment, the expression vector used may be the same (other than theprotein coding sequence) or different.

In a further embodiment, the one or more expression vectors encode allthree of the proteins comprising an amino acid sequence at least 80percent identical to the recited amino acid sequences (i.e.: SEQ IDNO:2, SEQ ID NO:4, and SEQ ID NO:6), or antigenic fragments thereof. Inthis embodiment, a single expression vector may encode all threeproteins, or antigenic fragments thereof. Alternatively, the compositionmay comprise two expression vectors, with one expression vector encodingone of the recited proteins, or antigenic fragments thereof, and theother expression vector encoding two of the recited proteins, orantigenic fragments thereof. In a further alternative of thisembodiment, the composition may comprise three expression vectors, witheach expression vector encoding one of the recited proteins, orantigenic fragments thereof. In each of these alternative embodiments,the multiple expression vectors used may be the same or different.Non-limiting examples of expression vectors encoding the one or morerecited proteins are provided herein. Based on the present disclosure,it is well within the level of those of skill in the art to prepareexpression vectors according to all embodiments of the invention.

The Cj0998c, Cj0588, and Cj0248 proteins are present in highly conservedvariants between different strains of C. jejuni, and thus the proteinsencoded by the one or more expression vectors can be at least 80%identical or similar (residues with similar properties, i.e.:hydrophobic, hydrophilic, etc.) over the full length of the recitedamino acid sequences, or antigenic fragments thereof. In various furtherembodiments, the proteins encoded by the one or more expression vectorsare at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100% percent identical over the full length of therecited amino acid sequence(s), or antigenic fragment thereof.

In one embodiment, the at least one polynucleotide encodes a proteinselected from the group consisting of the following, or an antigenicfragment thereof:

(a) SEQ ID NO: 7 (Cj0998c genus)MKK(I/V/F)(L/V)(V/A/L)S(V/I)(L/F)S(S/F)CLLASALSAVSFKEDSLK(I/V)SFEGYKTKDM(I/V)G(T/V/I/A)(K/R)GEFKNVEY(K/N)FSK(N/S)(I/T)KD(L/F)ASYLKGAKATI(K/E)PS(N/D)AFM(G/S)EG(N/L)D(I/V)ITNNITKVFFPALLG(D/N)(T/A)DIKVVFQD(V/A/M)I(A/V)GE-X1ITMDKKSTI(V/I)PLTYTIKD(N/D)KFEAKGQ(L/F)DLH(T/A)FKN(G/A)SKALKALSD(V/A)A(A/T/P)GHGGISWPLVDISFNALD(A/T/V)E wherein X1 is absent or is(SEQ ID NO: 135) NKGVISAK; (b) SEQ ID NO: 8: (Cj0588 genus)X1-(L/M)(D/N/E)LL(S/R)EIY(V/I)SRAALKLK(K/N)FLEEN(D/G/N)IE(I/V)(K/N)(H/Q/N)KNCLDIGSSTGGFVQILLEN(Q/K)ALKIT(A/T)LDVG(S/N)NQLH(P/S/L)(S/N)LR(V/A/T)NE(K/I)(I/V)IL(H/Y)EN(T/I)DLR(A/T/V)FKSEEKFE(L/F)(V/I)TCDVSFISL(I/V)NLLYY(I/V)(D/N)NLAL(K/R)EIILLFKPQFEVGKN(I/V)KRDKKGVLKD(D/G)(K/R)(A/V)ILKA(R/K)MDFEK(A/E)CAKL(G/S)W(L/F/I)LKNTQKS(S/C)IKG KEGNVEYFYYYIKNwherein X1 is absent or is SEQ ID NO: 136)M(R/I)(F/-)(D/-)FF(V/I)SKRL(N/D)ISRNKALELIE(N/S)EE(I/V)LLNGK(S/N)FKAS(F/C)DVKN(F/L)LENLKK(T/A/K)QDLN(P/L/S)E(D/E)(I/V)(L/Y)L(A/T/S)(N/D/K)(E/G) L(K/N); and(c) SEQ ID NO: 9 (Cj0248 genus)(M/-)I(G/-)DMNELLLKSVEVLPPLPDTVSKLRKYVSEANSNIETMKV(A/V)EIISSDPLMTAKLLQLANSPYYGFTREITTI(N/S)QVITLLG(V/I)GNIINIV(M/T)ADSI(R/K)D(N/S)FKIDVSPYGL(N/D)T(Q/K)(N/V)FL(K/R)(T/N)CN(E/D)EATFI(A/V/T)NWLNDEDKKLSHLLVPCAMLLRLGIVIFSNFLIQN(H/Y/F)(K/R)-X1wherein X1 is absent, or is (SEQ ID NO: 137 or 138)(D/E)K(D/E)FL(A/T)FLN(-/E/K)(-/T)K(N/S/I)EN(L/I)ALAENEFLGVDHISFLGFLL(H/Y)RWNFDD(V/I)LIESICFV(R/H)TPHAARE(K/E)VKKSAYALAITDHLF(A/T)PHDGSSPFN(A/V/ T)KAAVALL(K/E)EAK-X2;and wherein X2 is absent or is selected from the group consisting of(SEQ ID NO: 139) TQGINFDL(N/D)NLLSKLP(N/S)KAKENL(N/D)(K/E)ED and(SEQ ID NO: 140) NSRN.

In various further embodiments, the at least one polynucleotide encodesa protein selected from the group consisting of SEQ ID NOS: 10-106, oran antigenic fragment thereof. SEQ ID NOS, 44-82 are each Cj0998cprotein homologs from other C. jejuni strains; SEQ ID NOS, 83 to 106 areeach Cj0588 protein homologs from other C. jejuni strains; and SEQ IDNOS, 10 to 43 are each Cj0248 protein homologs from other C. jejunistrains.

The expression vectors may encode “antigenic portions” of the recitedprotein. As used herein, and “antigenic portion” is any fragment of 10or more contiguous amino acids in the recited amino acid sequence. Invarious further embodiments, the antigenic portion is any fragment ofany of the embodiments of the invention is 15, 20, 25, 30, 40, 50, 75,100, 125, 150, 175, 200, 225, 250, or 275 contiguous amino acids of therecited amino acid sequence.

The one or more isolated polynucleotides may be single or doublestranded DNA, RNA, genomic DNA, or cDNA. The one or more isolatedpolynucleotides may be any nucleic acids encoding the recited one ormore proteins, or antigenic fragments thereof. In one embodiment, theone or more isolated polynucleotides are one or more of SEQ ID NO:1(Cj0998c gene), SEQ ID NO:3 (Cj0588 gene), and SEQ ID NO:5 (Cj0248gene), or portions thereof encoding antigenic portions of the recitedproteins. It will be apparent to those of skill in the art, based on theteachings herein, what polynucleotide sequences will encode the recitedpolypeptides or antigenic fragments thereof.

As used herein, “isolated polynucleotides” are those that have beenremoved from their normal surrounding nucleic acid sequences in thegenome or in cDNA sequences. Such isolated nucleic acid sequences maycomprise additional sequences useful for promoting expression and/orpurification of the encoded protein, including but not limited to polyAsequences, modified Kozak sequences, and sequences encoding epitopetags, export signals, secretory signals, nuclear localization signals,and plasma membrane localization signals, as appropriate for a givenuse.

Any expression vector suitable for an intended use can be used in theimmunogenic compositions of the present invention. Such expressionvectors can be of any type known in the art, including but not limitedto plasmid and viral-based expression vectors. The construction ofexpression vectors for use in transfecting prokaryotic cells is alsowell known in the art, and thus can be accomplished via standardtechniques. (See, for example, Sambrook, Fritsch, and Maniatis, in:Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1989; Gene Transfer and Expression Protocols, pp. 109-128, ed. E.J. Murray, The Humana Press Inc., Clifton, N.J.), and the Ambion 1998Catalog (Ambion, Austin, Tex.). The expression vector must be replicablein the host organisms either as an episome or by integration into hostchromosomal DNA. In a preferred embodiment, the expression vectorcomprises a plasmid. However, the invention is intended to include otherexpression vectors that serve equivalent functions, such as viralvectors. Specifics of the expression vector will depend on the ultimatedesired use. Designing appropriate expression vectors for an intendeduse is well within the level of those of skill in the art based on theteachings herein.

Any suitable promoter may be used that can direct expression (i.e.: is“operatively linked”) of the encoded proteins. The term “promoter”includes any nucleic acid sequence sufficient to direct expression ofthe encoded protein(s), including inducible promoters, repressiblepromoters and constitutive promoters. If inducible, there are sequencespresent which mediate regulation of protein expression so that thepolynucleotide is transcribed only when an inducer molecule is present.Such cis-active sequences for regulated expression of an associatedpolynucleotide in response to environmental signals are well known tothe art. The expression vector may comprise any other control sequencesas may be suitable for an intended use. The control sequences need notbe contiguous with the nucleic acid sequences, so long as they functionto direct the expression thereof. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the nucleic acid sequences and the promoter sequence canstill be considered “operably linked” to the coding sequence. Other suchcontrol sequences include, but are not limited to, polyadenylationsignals, enhancers, termination signals, and ribosome binding sites.

The immunogenic compositions of the present invention may furthercomprise any other suitable components as may be useful for a givenpurpose. In various non-limiting embodiments, the compositions mayfurther comprise one or more expression vectors comprising at least onepolynucleotide encoding a protein selected from the group consisting ofproteins comprising an amino acid sequence at least 80 percent identicalto an amino acid sequence of SEQ ID NO:142 (Cj1534c protein; encoded,for example, by SEQ ID NO:141), SEQ ID NO:108 (1656c protein; encoded,for example, by SEQ ID NO:107), SEQ ID NO:110 (0428 protein; encoded,for example, by SEQ ID NO:109), SEQ ID NO:112 (0168c protein; encoded,for example, by SEQ ID NO:111), SEQ ID NO:114 (0427 protein; encoded,for example, by SEQ ID NO:113), SEQ ID NO:116 (Cj0113 protein; encoded,for example, by SEQ ID NO:115), SEQ ID NO:118 (Cj0982c protein; encoded,for example, by SEQ ID NO:117), SEQ ID NO:120 (Cj0921c protein; encoded,for example, by SEQ ID NO:119), SEQ ID NO:122 (Cj1259 protein; encoded,for example, by SEQ ID NO:121), SEQ ID NO:124 (Cj1339c protein; encoded,for example, by SEQ ID NO:123), SEQ ID NO:126 (Cj0034c protein; encoded,for example, by SEQ ID NO:125), SEQ ID NO:128 (Cj0404 protein; encoded,for example, by SEQ ID NO:127), SEQ ID NO:130 (Cj0365c protein; encoded,for example, by SEQ ID NO:129), SEQ ID NO:132 (Cj0755 protein; encoded,for example, by SEQ ID NO:131), and SEQ ID NO:134 (Cj0420 protein;encoded, for example, by SEQ ID NO:133), or antigenic fragments thereof.

The one or more expression vectors may be the same or different one ormore expression vectors that comprise the at least one polynucleotideencoding SEQ ID NO:2 (Cj0998c protein), SEQ ID NO:4 (Cj0588 protein),SEQ ID NO:6 (Cj0248 protein), or fragments thereof. All embodiments ofthe one or more expression vectors disclosed above apply equally forthese additional components. By way of non-limiting example, the one ormore expression vectors may be 1, 2, 3, or 4 additional vectors thatencode SEQ ID NO:142 (Cj1534c protein), SEQ ID NO:108 (1656c protein),SEQ ID NO:110 (0428 protein), SEQ ID NO:112 (0168c protein), and SEQ IDNO:114 (0427 protein), or antigenic fragments thereof. Those of skill inthe art will understand the variety of other combinations that can beemployed in accordance with the methods of the invention. Based on thepresent disclosure, it is well within the level of those of skill in theart to prepare expression vectors according to all embodiments of theinvention.

Similar to the immunogens recited above, the optional protein immunogensare present in highly conserved variants between different strains ofCampylobacter jejuni, and thus the proteins encoded by the one or moreexpression vectors can be at least 80% identical over the full length ofthe recited amino acid sequences, or antigenic fragments thereof. Invarious further embodiments, the proteins encoded by the one or moreexpression vectors are at least 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% percent identical over thefull length of the recited amino acid sequence(s), or antigenic fragmentthereof.

The immunogenic compositions of the present invention may be used forinducing an immune response by administration as naked DNA usingstandard methods, such as by parenteral delivery. Alternatively, theexpression vectors may comprise viral expression vectors, including butnot limited to a recombinant adeno-associated virus (AAV) gene deliveryvector. In this embodiment, the expression vector is bounded on the 5′and 3′ end by functional AAV inverted terminal repeat (ITR) sequences.By “functional AAV ITR sequences” is meant that the ITR sequencesfunction as intended for the rescue, replication and packaging of theAAV virion. Recombinant AAV (rAAV) virions encapsidating the expressionvectors of the invention may be produced using standard methodology. Inone embodiment, an AAV expression vector according to the invention isintroduced into a producer cell, followed by introduction of an AAVhelper construct, where the helper construct includes AAV coding regionscapable of being expressed in the producer cell and which complement AAVhelper functions absent in the AAV vector. This is followed byintroduction of helper virus and/or additional vectors into the producercell, wherein the helper virus and/or additional vectors provideaccessory functions capable of supporting efficient rAAV virusproduction. The producer cells are then cultured to produce rAAV. Thesesteps are carried out using standard methodology. Replication-defectiveAAV virions encapsulating the recombinant AAV vectors of the instantinvention are made by standard techniques known in the art using AAVpackaging cells and packaging technology. Examples of these methods maybe found, for example, in U.S. Pat. Nos. 5,436,146; 5,753,500,6,040,183, 6,093,570 and 6,548,286, expressly incorporated by referenceherein in their entirety. Further compositions and methods for packagingare described in Wang et al. (US 2002/0168342), also incorporated byreference herein in its entirety. Any suitable method for producingviral particles for delivery can be used.

In another embodiment, the one or more expression vectors are present ina carrier cell, including but not limited to an avirulent,non-Campylobacter bacterial carrier cell. Live bacterial vaccine“vectors” (i.e.: bacterial cells comprising immunogenic compositions)have been used successfully to elicit effective immune responses inorder to prevent infection. Recombinant attenuated bacterial celldelivered vaccines have been adapted to stably express protectiveantigens at high levels. They are capable of stimulating strong primaryhumoral, mucosal and lasting memory immune responses without significanttissue damage or other performance reducing effects. In variousnon-limiting embodiments, the bacterial carrier cell is an avirulentbacterial cell selected from the group consisting of attenuated L.monocytogenes, attenuated Salmonella spp., attenuated V. cholerae,attenuated Shigella spp., attenuated M. bovis BCG, attenuated Y.enterocolitica, attenuated B. anthracis, S. gordonii, Lactobacillusspp., and Staphylococcus spp. As used herein, “attenuated” means thatthe bacteria is reduced in causing disease symptoms in a host it isdelivered to compared to a non-attenuated bacterial vector. Suitableattenuated bacteria can be any species or strain that is or can besufficiently attenuated to allow for its non-pathological administrationto humans and/or animals in live and/or dead form. In one embodiment, anattenuated Salmonella species is used. In exemplary embodiments,Salmonella that can be used include, but are not limited to Salmonellaenterica strains selected from the group consisting of S. Typhimurium,S. Enteriditis, S. Heidelberg, S. Gallinarum, S. Hadar, S. Agona, S.Kentucky, S. Typhi, S. Paratyphi and S. Infantis. S. Typhimurium isespecially useful for vaccination purposes because the genome sequenceis fully characterized and many animal studies confirm its safe medicaluse. Recombinant attenuated Salmonella vaccines (RASVs) have beenconstructed to deliver antigens from other pathogens to induce immunityto those pathogens in vaccinated hosts; see, for example, Curtiss etal., Crit Rev Immunol. 2010; 30(3):255-70; 2010; Qiu et al., J.Virological Methods 188:108; Strugnell et al., Infect. Immunol. 1992,60:3994; Layton et al., Clinical and Vaccine Immunology March 2011,449-454; Al-Ojali et al., Microbial Pathogenesis 52:326 (2012); and(Wyszynska et al., 2004) Wyszyńska et al. (2004). In one embodiment, theRASV comprises attenuating mutations in the pmi (mannose-6-phosphateisomerase), fur (ferric uptake regulator) and crp (cAMP regulatoryprotein) genes (see, for example, (Li et al., PNAS 106:592-597 2009,Curtiss et al., 2009) and U.S. Pat. No. 8,133,493. In anotherembodiment, the RASV comprises the χ9992 vector disclosed in U.S. Pat.No. 8,133,493. In another embodiment, the RASV is one that iscommercially available, such as Megan®Vac1 (Lohman Animal Health, US).

Attenuated bacterial cells can be transfected with the one or moreexpression vectors using standard techniques in the art.

In a second aspect, the present invention provides an immunogeniccomposition, comprising

(a) one or more isolated proteins selected from the group consisting ofproteins comprising an amino acid sequence at least 80 percent identicalto SEQ ID NO:2 (Cj0998c protein), SEQ ID NO:4 (Cj0588 protein), and SEQID NO:6 (Cj0248 protein), or antigenic portions thereof; and

(b) a pharmaceutically acceptable carrier.

As disclosed above for the first aspect of the invention, theimmunogenic compositions of the second aspect of the invention can beused, for example for stimulating an immune response in subjects at riskof C. jejuni infection or colonization, including but not limited tovertebrates such as chickens, turkeys, cattle, sheep, pigs, and humans.

In one embodiment, the immunogenic composition comprises one of therecited proteins, or antigenic fragments thereof. In another embodiment,the immunogenic composition comprises two of the proteins comprising anamino acid sequence at least 80 percent identical to the recited aminoacid sequences (i.e.: SEQ ID NO:2 and SEQ ID NO:4; SEQ ID NO:2 and SEQID NO:6; or SEQ ID NO:6 and SEQ ID NO:8), or antigenic fragmentsthereof. In a further embodiment, the immunogenic composition comprisesall three of the proteins comprising an amino acid sequence at least 80percent identical to the recited amino acid sequences (i.e.: (i.e.: SEQID NO:2, SEQ ID NO:4, and SEQ ID NO:6), or antigenic fragments thereof.

The Cj0998c, Cj0588, and Cj0248 proteins are present in highly conservedvariants between different strains of C. jejuni, and thus the proteinscan be at least 80% identical over the full length of the recited aminoacid sequences, or antigenic fragments thereof. In various furtherembodiments, the proteins are at least 81, 82, 83, 84, 85, 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% percent identicalover the full length of the recited amino acid sequence(s), or antigenicfragment thereof.

In one embodiment, the at least one protein is selected from the groupconsisting of the following, or an antigenic fragment thereof:

(a) SEQ ID NO: 7 (Cj0998c genus)MKK(I/V/F)(L/V)(V/A/L)S(V/I)(L/F)S(S/F)CLLASALSAVSFKEDSLK(I/V)SFEGYKTKDM(I/V)G(T/V/I/A)(K/R)GEFKNVEY(K/N)FSK(N/S)(I/T)KD(L/F)ASYLKGAKATI(K/E)PS(N/D)AFM(G/S)EG(N/L)D(I/V)ITNNITKVFFPALLG(D/N)(T/A)DIKVVFQD(V/A/M)I(A/V)GE-X1ITMDKKSTI(V/I)PLTYTIKD(N/D)KFEAKGQ(L/F)DLH(T/A)FKN(G/A)SKALKALSD(V/A)A(A/T/P)GHGGISWPLVDISFNALD(A/T/V)E wherein X1 is absent or is(SEQ ID NO: 135) NKGVISAK; (b) SEQ ID NO: 8: (Cj0588 genus)X1-(L/M)(D/N/E)LL(S/R)EIY(V/I)SRAALKLK(K/N)FLEEN(D/G/N)IE(I/V)(K/N)(H/Q/N)KNCLDIGSSTGGFVQILLEN(Q/K)ALKIT(A/T)LDVG(S/N)NQLH(P/S/L)(S/N)LR(V/A/T)NE(K/I)(I/V)IL(H/Y)EN(T/I)DLR(A/T/V)FKSEEKFE(L/F)(V/I)TCDVSFISL(I/V)NLLYY(I/V)(D/N)NLAL(K/R)EIILLFKPQFEVGKN(I/V)KRDKKGVLKD(D/G)(K/R)(A/V)ILKA(R/K)MDFEK(A/E)CAKL(G/S)W(L/F/I)LKNTQKS(S/C)IKG KEGNVEYFYYYIKNwherein X1 is absent or is SEQ ID NO: 136)M(R/I)(F/-)(D/-)FF(V/I)SKRL(N/D)ISRNKALELIE(N/S)EE(I/V)LLNGK(S/N)FKAS(F/C)DVKN(F/L)LENLKK(T/A/K)QDLN(P/L/S)E(D/E)(I/V)(L/Y)L(A/T/S)(N/D/K)(E/G) L(K/N); and(c) SEQ ID NO: 9 (Cj0248 genus)(M/-)I(G/-)DMNELLLKSVEVLPPLPDTVSKLRKYVSEANSNIETMKV(A/V)EIISSDPLMTAKLLQLANSPYYGFTREITTI(N/S)QVITLLG(V/I)GNIINIV(M/T)ADSI(R/K)D(N/S)FKIDVSPYGL(N/D)T(Q/K)(N/V)FL(K/R)(T/N)CN(E/D)EATFI(A/V/T)NWLNDEDKKLSHLLVPCAMLLRLGIVIFSNFLIQN(H/Y/F)(K/R)-X1wherein X1 is absent, or is (SEQ ID NO: 137 or 138)(D/E)K(D/E)FL(A/T)FLN(-/E/K)(-/T)K(N/S/I)EN(L/I)ALAENEFLGVDHISFLGFLL(H/Y)RWNFDD(V/I)LIESICFV(R/H)TPHAARE(K/E)VKKSAYALAITDHLF(A/T)PHDGSSPFN(A/V/ T)KAAVALL(K/E)EAK-X2;and wherein X2 is absent or is selected from the group consisting of(SEQ ID NO: 139) TQGINFDL(N/D)NLLSKLP(N/S)KAKENL(N/D)(K/E)ED and(SEQ ID NO: 140) NSRN.

In various further embodiments, the at least one polynucleotide encodesa protein selected from the group consisting of SEQ ID NOS: 10-106, oran antigenic fragment thereof. SEQ ID NOS, 44-82 are each Cj0998cprotein homologs from other C. jejuni strains; SEQ ID NOS, 83-106 areeach Cj0588 protein homologs from other C. jejuni strains; and SEQ IDNOS, 10-43 are each Cj0248 protein homologs from other C. jejunistrains.

The immunogenic composition of any embodiment of this second aspect ofthe invention may comprise “antigenic portions” of the recited proteins.As used herein, and “antigenic portion” is any fragment of 10 or morecontiguous amino acids in the recited amino acid sequence. In variousfurther embodiments, the antigenic portion is any fragment of 15, 20,25, 30, 40, 50, 75, 100, 125, 150, 175, 200, 225, 250, or 275 contiguousamino acids of the recited amino acid sequence.

The immunogenic compositions of the second aspect of the invention maycomprise proteins modified in any suitable way. In one embodiment, theprotein component(s) of the composition are treated to extend in vivohalf-life by, for example, such as by PEGylation, HESylation,PASylation, or glycosylation. The proteins may also be glycosylated asdeemed appropriate, using standard techniques in the art. In anotherembodiment, those protein components in the immunogenic compositionsthat possess N-glycosylation sequences (NXS or NXT) may be glycosylated,to help further stimulate the immune response.

The immunogenic compositions of the second aspect of the invention mayfurther comprise any other suitable components as may be useful for agiven purpose. In various non-limiting embodiments, the compositions mayfurther comprise one or more additional proteins selected from the groupconsisting of proteins comprising an amino acid sequence at least 80%identical to SEQ ID NO:142 (Cj1534c protein), SEQ ID NO:108 (Cj1656cprotein), SEQ ID NO:110 (Cj0428 protein), SEQ ID NO:112 (Cj0168cprotein), SEQ ID NO:114 (Cj0427 protein), SEQ ID NO:116 (Cj0113protein), SEQ ID NO:118 (Cj0982c protein), SEQ ID NO:120 (Cj0921cprotein), SEQ ID NO:122 (Cj1259 protein), SEQ ID NO:124 (Cj1339cprotein), SEQ ID NO:126 (Cj0034c protein), SEQ ID NO:128 (Cj0404protein), SEQ ID NO:130 (Cj0365c protein), SEQ ID NO:132 (Cj0755protein), and SEQ ID NO:134 (Cj0420 protein), or antigenic fragmentsthereof. Similar to the immunogens recited above, the optional proteinimmunogens are present in highly conserved variants between differentstrains of Campylobacter jejuni, and thus the proteins can be at least80% identical over the full length of the recited amino acid sequences,or antigenic fragments thereof. In various further embodiments, theproteins encoded by the one or more expression vectors are at least 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99,or 100% percent identical over the full length of the recited amino acidsequence(s), or antigenic fragment thereof.

In another embodiment, the immunogenic compositions of the second aspectof the invention may further comprise a C. jejuni pilus protein asdescribed in WO 2008/008092, incorporated by reference herein in itsentirety. The pilus protein described in WO 2008/008092 was shown tostimulate an immune response against C. jejuni, and thus it is suitablefor inclusion in the immunogenic compositions of the second aspect ofthe present invention. Methods for isolating the C. jejuni pilus proteinare described in WO 2008/008092.

The immunogenic compositions of the invention may comprise any suitableamount/dosage of the composition as determined most appropriate. In oneembodiment, the immunogenic composition comprises about 10⁶-10¹⁰avirulent bacterial cells per dose. In another embodiment where thecomposition comprises immunogenic proteins, the composition may compriseabout 0.1 ug/kg-100 mg/kg body weight of the proteins; alternatively, itmay be 0.5 ug/kg to 50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10mg/kg body weight of the proteins.

The immunogenic compositions of the present invention (i.e.: anyembodiment or combination of embodiments of the first and second aspectsof the invention) can be formulated by any of the means known in theart. The immunogenic compositions are typically formulated as apharmaceutical composition, such as those disclosed above, and can beformulated for administration via any suitable route, including orally,as injectables, parentally, by inhalation spray, intranasally, rectally,mucosally, topically, or for administration by oral gavage or ad libitumfeeding, for example, in drinking water, either as liquid solutions orsuspension, in dosage unit formulations containing conventionalpharmaceutically acceptable carriers, adjuvants, and vehicles. The termparenteral as used herein includes, subcutaneous, intravenous,intra-arterial, intramuscular, intrasternal, intratendinous,intraspinal, intracranial, intrathoracic, infusion techniques orintraperitoneally. Solid forms suitable for solution in, or suspensionin, liquid prior to injection or other administration may also beprepared. The compositions may also, for example, be emulsified, or theencapsulated in liposomes or microparticles. The immunogeniccompositions may also be present in and/or expressed by transgenicplants.

The immunogenic compositions according to the present invention mayfurther comprise any suitable adjuvant. Immunological adjuvants ingeneral comprise substances that boost the immune response of the hostin a nonspecific manner. A number of different adjuvants are known inthe art. Examples of adjuvants are Freunds Complete and Incompleteadjuvant, vitamin E, non-ionic block polymers and polyamines such asdextransulphate, carbopol and pyran, oligopeptide, emulsifiedparaffin-Emulsigen™ (MVP Labs, Ralston, Nebr.), L80 adjuvant containingaluminum hydroxide (Reheis, N.J.), Quil A™ (Superphos); surface activesubstances such as Span™, Tween™, hexadecylamine, lysolecitin,methoxyhexadecylglycerol and saponins; peptides such asmuramyldipeptides, dimethylglycine, and tuftsin; immune-stimulatingcomplexes (ISCOMS), mineral oil e.g. Bayol or Markol, vegetable oils oremulsions thereof, aluminum hydroxide;N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-s-n-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria: monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. In embodiments designed for mucosal administration, theimmunogenic compositions may further comprise an adjuvant, including butnot limited to such as the nontoxic cholera toxin B subunit (SigmaChemical Company, St. Louis, Mo.) and organometallopolymers includinglinear, branched or cross-linked silicones which are bonded at the endsor along the length of the polymers to the particle or its core. Suchpolysiloxanes can vary in molecular weight from about 400 up to about1,000,000 daltons; the preferred length range is from about 700 to about60,000 daltons. Suitable functionalized silicones include(trialkoxysilyl) alkyl-terminated polydialkylsiloxanes andtrialkoxysilyl terminated polydialkylsiloxanes, for example,3-(triethyoxysilyl) propyl terminated polydimethylsiloxane. See U.S.Pat. No. 5,571,531, incorporated by reference herein. Phosphazenepolyelectrolytes can also be incorporated into immunogenic compositionsfor mucosal administration (See e.g., U.S. Pat. No. 5,562,909).

The immunogenic compositions according to the present invention may alsocomprise preservatives such as sodium azide, thimersol, gentamicin,neomycin, and polymyxin.

The immunogenic compositions may be mixed with excipients or carrierswhich are pharmaceutically acceptable and compatible with the proteinimmunogen(s) to be used. Suitable excipients include, but are notlimited to, water, saline, dextrose, glycerol, ethanol, or the like andcombinations thereof. Such an immunogenic composition can easily beprepared by admixing the protein with a pharmaceutically acceptablecarrier. A pharmaceutically acceptable carrier is understood to be acompound that does not adversely affect the health of the animal to bevaccinated, at least not to the extent that the adverse effect is worsethan the effects seen when the animal is not vaccinated. Apharmaceutically acceptable carrier can be e.g. sterile water or asterile physiological salt solution. In a more complex form, the carriercan e.g. be a buffer.

The immunogenic composition may further comprises stabilizers, e.g. toprotect degradation-prone components from being degraded, to enhance theshelf-life of the composition, or to improve freeze-drying efficiency.Useful stabilizers include, without limitation, SPGA, skimmed milk,gelatin, bovine or other serum albumin, carbohydrates e.g. sorbitol,mannitol, trehalose, starch, sucrose, dextran or glucose, proteins suchas albumin or casein or degradation products thereof, and buffers, suchas alkali metal phosphates. Where an albumin is used, it is desirablyfrom the same species as the animal (or human) to which the immunogeniccomposition containing it will be administered. Freeze-drying is anefficient method for conservation. Freeze-dried material can be storedstable for many years. Storage temperatures for freeze-dried materialmay well be above zero degrees, without being detrimental to thematerial. Freeze-drying can be done according to all well-known standardfreeze-drying procedures. However, the immunogenic compositions of theinvention may be stored in any suitable manner. For example, theimmunogenic compositions could be lyophilized or otherwise stabilizedand stored in food or water for delivery.

In an exemplary embodiment when the immunogenic compositions aredesigned for administration to a non-human subject, such as chickens,turkeys, birds, sheep, pigs, cattle, dogs, or cats, the immunogeniccomposition is formulated for mucosal administration, such as byadmixing of the composition with drinking water or food, or admixing foruse as a spray, to mist over the animals to uptake the immunogeniccomposition during grooming behavior. However, any suitable method ofadministration can be used for any subject. Thus, in various embodimentsthe immunogenic compositions are formulated for intraocular, intranasal,or transdermal administration.

In a third aspect, the present invention provides methods forstimulating an immune response against Campylobacter, comprisingadministering to a subject an effective amount of the immunogeniccomposition according to any embodiment or combination of embodiments ofthe first or second aspects of the present invention to generate animmune response against Campylobacter.

In a fourth aspect, the present invention provides methods for reducingCampylobacter intestinal colonization in a subject, comprisingadministering an amount effective of the immunogenic compositionaccording to any embodiment or combination of embodiments of the firstor second aspects of the present invention to reduce Campylobacterintestinal colonization in the subject.

As disclosed herein, the inventors have identified three novel putativevirulence genes (Cj0248, Cj0588, and Cj0998c) from C. jejuni encodingnovel proteins from the outer-membrane of the bacterium. The inventorshave further discovered that each of the Cj0998c protein, the Cj0588protein, and the Cj0248 protein are potent immunogens for stimulating aneffective immune response against Campylobacter jejuni (“C. jejuni”).For example, as disclosed in detail herein, two separate vaccinationtrials of chickens with a vector expressing the Cj0988c proteindemonstrated reduced numbers of C. jejuni in birds after challenge anaverage of 2.5 logs CFU (geomean 3 logs) when compared to the cecalnumbers of non-vaccinated control birds. Furthermore, vaccination trialsdemonstrated a significant reduction (1-4 logs) (1 log with heterologousstrain and 4 logs with homologous strain) of C. jejuni in cecal contentsof chickens vaccinated with vectors expressing the Cj0588 protein andchallenged. Each of the proteins was initially isolated from anouter-membrane (OMP) extraction of a C. jejuni biofilm.

Campylobacteriosis is a food-borne disease primarily generally caused byC. jejuni. The major risk factor in acquiring the disease is thehandling and consumption of poultry. However, the epidemiology ofpoultry colonization with Campylobacter such as C. jejuni is extremelycomplex. Birds become colonized within 14 days of hatching, spreadingthe infection throughout the flock by the end of the grow-out period.Strain differences, based on subtyping and/or genotyping assays, existbetween and within flocks. Although some flocks remainCampylobacter-free, most flocks have 50-100% of the birds colonized bygrow-out. Nevertheless, broilers (i.e., chickens of either gender thatwill be slaughtered for meat at about 5 to 8 weeks old depending onweight) become contaminated with Campylobacter, such as C. jejuni andalthough this microorganism acts as normal flora in the chicken,undercooked chicken is a primary vehicle for transmission ofCampylobacter, such as C. jejuni to humans and remains a significantpublic health concern.

The methods may be used on any suitable subject at risk of Campylobacterinfection, including but not limited to vertebrates such as chickens,turkeys, birds, cattle, sheep, pigs, dogs, cats, and humans. In onenon-limiting embodiment, a human subject may be anyone that consumeschicken, beef, turkey, or pork. In another embodiment, a human subjectmay be one that works with animals (i.e.: farm workers, workers atslaughterhouses and meat processing plants, etc.) such as chickens,turkeys, cattle, sheep, and pigs.

In another embodiment, the subject is a feed animal such as chickens,turkeys, cattle, sheep, and pigs. In a preferred embodiment, the subjectis a chicken. In these embodiments, the feed animal may be of anysuitable age. Birds become colonized with C. jejuni within 14 days ofhatching, spreading the infection throughout the flock by the end of thegrow-out period. Thus, in one embodiment, the methods are initiallycarried out by about 14 days after hatching. It will be understood bythose of skill in the art that additional booster administrations may bedesirable after the initial administration; such booster administrationscan be carried out at any suitable time, such as by about 21 days afterhatching.

In another non-limiting embodiment where the subject is a feed animal,the methods are carried out before slaughtering. However, it will beunderstood that the methods can be used on chickens or turkeys at anysuitable time, as appropriate for a given use.

Campylobacteriosis is currently one of the most common bacterialfood-borne diseases in humans in the U.S. and is responsible for causingan estimated 1.3 million cases annually in the U.S., often accompaniedby acute gastroenteritis. The infectious dose in the development of thedisease is variable and ranges from 500 to 10⁶ organisms. Variation ininfectious dose is thought to be due to either individual susceptibilityor to the relative virulence of the organism. The incubation period isone to seven days with clinical symptoms including fever, severeabdominal cramps, and a watery diarrhea or a dysentery-like syndrometypical of shigellosis. The disease is usually self-limiting, lastingfrom two to seven days but occasionally is fatal (120-360 deaths peryear), mainly in infants and young adults. Serious complications such asarthritis occur in an estimated 1-5% of cases and Guillain-BarreSyndrome, a form of neuromuscular paralysis occurs at a rate of 1 per1,000 patients.

Conversely, Campylobacter, such as C. jejuni, colonizes poultry as acommensal, that is, without producing any overt signs of disease.Similarly, Campylobacter, such as C. jejuni may colonize other feedanimals as a commensal as well. However, infected cattle may suffer fromdiarrhea, weight loss, and suffer from fever and increased heart rate.

The methods of the third and fourth aspects of the invention may be usedto stimulate an immune response against or to reduce intestinalcolonization of any species of Campylobacter. In preferred embodiments,the methods of the third and fourth aspects of the invention are used tostimulate an immune response against or to reduce intestinalcolonization of varied species of Campylobacter including but notlimited to C. jejuni, C. coli, C. lari and/or C. upsaliensis.

As used herein, methods for “stimulating an immune response” result inone or more effects (e.g., maturation, proliferation, direct- orcross-presentation of antigen, gene expression profile) on cells ofeither the innate or adaptive immune system. For example, the immuneresponse may involve, effect, or be detected in innate immune cells suchas, for example, dendritic cells, monocytes, macrophages, natural killercells, and/or granulocytes (e.g., neutrophils, basophils oreosinophils). The immune response may also involve, effect, or bedetected in adaptive immune cells including, for example, lymphocytes(e.g., T cells and/or B cells). The immune response may be observed bydetecting such involvement or effects including, for example, thepresence, absence, or altered (e.g., increased or decreased) expressionor activity of one or more immunomodulators. The immune response maystimulate a de novo or previously undetected antibody response, orenhance or suppress an existing response against the immunogen by, forexample, causing an increased antibody response (e.g., amount ofantibody, increased affinity/avidity) or an increased cellular response(e.g., increased number of activated T cells, and/or increasedaffinity/avidity of T cell receptors. In certain embodiments, the immuneresponse may be protective, meaning that the immune response may becapable of preventing initiation or continued infection of or growthwithin a host and/or by eliminating C. jejuni from the host. In someinstances, elimination of an agent from the host may mean that themethod is therapeutic, in that the method is used to treat a subjectalready infected with C. jejuni. When the method is therapeutic, themethod may comprise treating a C. jejuni infection, wherein “treating”means accomplishing one or more of the following: (a) reducing theseverity of the infection; (b) limiting or preventing development ofsymptoms characteristic of the infection; (c) inhibiting worsening ofsymptoms characteristic of the infection; (d) limiting or preventingrecurrence of the disorder(s) in subjects that have previously had theinfection; and (e) limiting or preventing recurrence of symptoms insubjects that were previously symptomatic for the infection.

As used herein, “reducing Campylobacter intestinal colonization” meansreducing a level of intestinal colonization that would be observed inthe subject in the absence of administering the one or more immunogeniccompositions of the invention. Any level of reduction is beneficial inreducing Campylobacter transmission, for example, from poultry to ahuman consuming the poultry. In one embodiment, the reduction comprisesat least 10% reduction in intestinal colonization compared to intestinalcolonization in the absence of treatment; in various furtherembodiments, the intestinal colonization is reduced by at least 20%,25%, 50%, 75%, 80%, 85%, 90%, 95%, or more compared to intestinalcolonization in the absence of treatment. Techniques for quantifying alevel of Campylobacter intestinal colonization are well known in the artand include, but are not limited to, examining cecal or fecal levels ofCampylobacter. Techniques for determining fecal contamination ofCampylobacter in production to quantify the level of Campylobacterorganisms from a carcass rinse of feed animals, such as poultry, atvarious points of processing, such as after chilling are alsoestablished.

In preferred embodiments of the third and fourth aspects of theinvention, the one or more expression vectors are present in a carriercell, including but not limited to a non-Campylobacter bacterial carriercell. In various non-limiting embodiments, the bacterial carrier cell isan avirulent bacterial cell selected from the group consisting ofattenuated L. monocytogenes, attenuated Salmonella spp., attenuated V.cholerae, attenuated Shigella spp., attenuated M. bovis BCG, attenuatedY. enterocolitica, attenuated B. anthracis, S. gordonii, Lactobacillusspp., and Staphylococcus spp. In one embodiment, an attenuatedSalmonella species is used. In exemplary embodiments, Salmonella thatcan be used include, but are not limited to Salmonella enterica strainsselected from the group consisting of S. Typhimurium, S. Enteriditis, S.Heidelberg, S. Gallinarum, S. Hadar, S. Agona, S. Kentucky, S. Typhi, S.Paratyphi and S. Infantis. S. Typhimurium is especially useful forvaccination purposes because the genome sequence is fully characterizedand many animal studies confirm its safe medical use. Recombinantattenuated Salmonella vaccines (RASVs) have been constructed to deliverantigens from other pathogens to induce immunity to those pathogens invaccinated hosts; see, for example, Curtiss et al., Crit Rev Immunol.2010; 30(3):255-70; 2010; Qiu et al., J. Virological Methods 188:108;Strugnell et al., Infect. Immunol. 1992, 60:3994; Layton et al.,Clinical and Vaccine Immunology March 2011, 449-454; Al-Ojali et al.,Microbial Pathogenesis 52:326 (2012); and Wyszyńska et al. (2004). Inone embodiment, the RASV comprises attenuating mutations in the pmi, furand crp genes and U.S. Pat. No. 8,133,493. In another embodiment, theRASV comprises the χ9992 vector disclosed in U.S. Pat. No. 8,133,493. Inanother embodiment, the RASV is one that is commercially available, suchas Megan®Vac1 (Lohman Animal Health, US).

The immunogenic compositions can be administered via any suitable route,including orally, as injectables, parentally, by inhalation spray,intranasally, rectally, mucosally, topically, or for administration byoral gavage or ad libitum feeding, for example, in drinking water,either as liquid solutions or suspension, in dosage unit formulationscontaining conventional pharmaceutically acceptable carriers, adjuvants,and vehicles. The term parenteral as used herein includes, subcutaneous,intravenous, intra-arterial, intramuscular, intrasternal,intratendinous, intraspinal, intracranial, intrathoracic, infusiontechniques or intraperitoneally. Solid forms suitable for solution in,or suspension in, liquid prior to injection or other administration mayalso be prepared. The compositions may also, for example, be emulsified,or the encapsulated in liposomes.

In an exemplary embodiment when the subject is a non-human subject, suchas chickens, turkeys, birds, sheep, pigs, cattle, dogs, or cats, theimmunogenic composition are administered orally or mucosally, such as byadmixing of the composition with drinking water or food, or admixing foruse as a spray, to mist over the animals to uptake the immunogeniccomposition during grooming behavior. In exemplary embodiments where thesubject is a human, the immunogenic compositions are administered orallyor mucosally. However, any suitable method of administration can be usedfor any subject.

The immunogenic compositions are administered in a manner compatiblewith the dosage formulation, and in such amount and manner as will beprophylactically and/or therapeutically effective, according to what isknown to the art. The quantity to be administered depends on the subjectto be treated, the functional capacity of the subject's immune system,the degree of protection desired, and other factors. Precise amounts ofthe active ingredient required to be administered may depend on thejudgment of the individual administering the immunogenic compositionsand may be peculiar to each individual, but such a determination iswithin the level of those of skill in the art based on the teachingsherein. In one embodiment, about 10⁶-10¹⁰ avirulent bacterial cells perdose can be administered. In another embodiment where immunogenicproteins are administered, a suitable dosage range may, for instance, be0.1 ug/kg-100 mg/kg body weight; alternatively, it may be 0.5 ug/kg to50 mg/kg; 1 ug/kg to 25 mg/kg, or 5 ug/kg to 10 mg/kg body weight.

The immunogenic compositions may be administered in a single dose or inmultiple dosages as determined most appropriate, such as a two doseschedule, for example two to eight weeks apart; or a multiple doseschedule or in combination with other vaccines. A multiple dose scheduleis one in which a primary course of vaccination may include 1 to 10 ormore separate doses, followed by other doses administered at subsequenttime intervals as required to maintain and/or reinforce the immuneresponse, e.g., at 1 to 4 months for a second dose, and if needed, asubsequent dose(s) after several months.

Example 1 Immunogenic Compositions Based on Cj0988c

The DNA and protein sequence of Cj0988c are provided in SEQ ID NO:1 andSEQ ID NO:2, respectively.

Salmonella Typhimurium Vector χ9992:

The vector used was a Salmonella Typhimurium vector χ9992 (Curtiss etal., 2009) which contains eight mutations, with three of the mutationsattenuating: the pmi, fur, and crp genes.

-   1) Δpmi-2426-Eliminates phosphomannose isomerase which ceases LPS-O    antigen synthesis in the absence of mannose;-   2) Δgmd-fcl-Reduces biofilm formation and prevents the formation of    colonic acid which aids in the survival of asdA mutants;-   3) ΔasdA27-Deletion of a gene encoding an enzyme necessary for    diaminopimelic acid (DAP). Strain requires supplemental DAP until    the introduction of the plasmid. Eliminates need for antibiotic    resistance markers;-   4) ΔP_(fur77)::TT araC P_(BAD)fur::TT araC P_(BAD)c2 ΔPcrp527::TT    araC P_(BAD)crp—Allows fur and crp gene expression only in the    presence of arabinose, producing pYA3493 (FIG. 1), a strain which is    maximally invasive prior to display of attenuated phenotype    following cell division;-   5) ΔrelA198::araCP_(BAD)laclTT—eliminates relA gene to uncouple    growth from protein synthesis and provides arabinose-dependent    synthesis of the LacI repressor to confer regulated delayed in vivo    synthesis of recombinant proteins, and-   6) ΔaraE25 ΔaraBAD23—Eliminates the ability of Salmonella to    metabolize arabinose. Allows retention of arabinose in the cytoplasm    without use.

The vector plasmid pYA3493 contains the asd gene to compliment thechromosomal ΔasdA27 mutation and ensure that the plasmid will not belost in vivo. The plasmid also fuses the expressed product to aβ-lactamase signal sequence for periplasmic secretion of the protein.Finally, the plasmid has a strong promoter (−35 region of trppromoter/−10 lac) in P_(trc) (FIG. 1). Use of this plasmid does notconfer antibiotic resistance to the Salmonella vaccine.

The vector that we used for the amplification of the plasmid with theinserted C. jejuni gene is E. coli χ6212. This strain is Asd− allowingamplification of the plasmid, which is then extracted and electroporatedinto Salmonella χ9992.

Gene Cj0998c expresses a hypothetical protein that is part of the outermembrane of the bacterium. Gene Cj0998c was cloned individually intoplasmid pYA3493 and expressed from the Salmonella vector χ9992.

A. Cloning of the Cj0998c Gene into the Salmonella Vector

The Salmonella strain (χ9992) used as the vector has three differentmutations which results in its attenuation. The strain is safe andimmunogenic and acts as a phenotypically wild-type strain at the time ofimmunization but becomes attenuated after colonization of host tissues.The plasmid contains the asd gene which provides a marker for laboratorydifferentiation using media not containing DAP to growplasmid-containing strains. Since this gene is required for the survivalof the vector without DAP, the plasmid is maintained in the vectorduring in vivo colonization. Gene Cj0998c was cloned into Salmonellaχ9992. Essentially, primers to each gene were designed and the geneamplified using PCR. Each gene product was inserted into plasmid pYA3493(asd+), cloned into E. coli χ6212 (asd−) and cultured for amplification.Following growth, the plasmid was extracted from the E. coli strain,cloned into the Salmonella Typhimurium vector, and the transformantsplated on LB media minus DAP, cultured at 37° C. on LB agar, harvested,and stored frozen at −80° C. Confirmation that the gene was present inthe vector was accomplished through a plasmid extraction and sequenceanalysis of genes following PCR and gel electrophoresis.

B. Salmonella Growth In Vitro

The Salmonella vector expressing the Cj0998c protein and the Salmonellaempty vector were grown in Luria Bertani (LB) broth supplemented with0.1% glucose, 0.05% mannose, and 0.1% arabinose for 24 h at 37° C. withshaking. Following incubation, the overnight cultures were pelleted andre-suspended in PBS to a final concentration of 1×10¹⁰ CFU/ml.

C. Vaccination of Poultry

Vaccination of chickens, twice, with the vector expressing the Cj0988cprotein reduced cecal numbers of C. jejuni in birds an average of 2.5logs CFU (geomean of 3.0 logs CFU) when compared to the cecal numbers ofnon-vaccinated control birds (Table 1). One outlier of 1.00E+07(highlighted cell, Table 3) was present in 31 birds examined. Cornish XRock chickens were obtained from McMurray Hatchery (IA) and randomlyassigned to isolated groups. The chickens were screened for the fecalpresence of C. jejuni and/or Salmonella. The chickens were vaccinatedorally, at days 10 and 16, with 1 ml of the Salmonella vector expressingthe Cj0988 protein (˜10¹⁰ CFU/ml) or the Salmonella empty vector andchallenged with 10⁵ CFU/ml of C. jejuni strain NCTC11168 10 days afterthe second vaccination. Prior to vaccination and challenge, chickenswere fasted for 8 hours. Food was returned one hour postvaccination/challenge. Aside from this period, food and water wereavailable ad libitum. The chickens were necropsied 10 days afterchallenge infection. Cecal contents were ten-fold diluted and directplated onto modified Campy Cefex agar plates incubated for 48 h at 10%CO₂ microaerophilic conditions for enumeration of Campylobacter.

TABLE 1 Combined data of C. jejuni vaccination trials 1 & 2 C. jejuniNCTC11168 Challenge-Trials 1&2 Cj0998c Control-EV Controls Average6.11E+05 6.75E+07 1.15E+08 Geomean 2.60E+04 4.41E+07 4.83E+07

TABLE 2 Vaccination Trial 1 - Ten day old chickens vaccinated twice withthe vector expressing gene Cj0998c, or the Salmonella empty vector only(Control-EV), or non- vaccinated controls C. jejuni NCTC11168Challenge - Trial 1 Cj0998c Control-EV Controls 1.0E+03 1.0E+07 3.0E+087.0E+04 1.2E+07 6.0E+07 7.4E+04 1.0E+08 1.0E+07 8.2E+04 2.0E+08 1.0E+071.0E+04 7.0E+07 2.0E+08 2.0E+04 9.0E+07 2.0E+07 3.0E+04 1.0E+08 4.0E+082.0E+03 1.0E+08 1.2E+08 6.0E+04 2.0E+07 1.0E+07 3.0E+04 1.0E+07 2.0E+07Average 3.79E+04  7.12E+07  1.15E+08  Geomean 1.98E+04  4.45E+07 4.83E+07 

TABLE 3 Vaccination Trial 2—Ten day old chickens vaccinated twice withthe vector expressing gene Cj0998c (two groups), or the Salmonella emptyvector only (Control-EV) C. jejuni NCTC11168 Challenge—Trial 2 Cj0998cCj0998c Control-EV 1.40E+04 2.00E+05 5.00E+07 3.20E+03 1.70E+05 3.00E+077.00E+04 3.00E+04 2.00E+07 1.00E+02 2.70E+05 1.70E+08 7.00E+04 3.00E+053.00E+07 9.00E+03 2.40E+04 1.05E+04 1.50E+04 1.00E+00 5.00E+06 6.00E+032.30E+06 3.00E+03  1.0E+07 6.00E+04 Average 2.23E+04 1.83E+06 6.00E+07Geomean 3.78E+03 2.84E+03 4.33E+07

Example 2 Immunogenic Compositions Based on Cj0588 and Cj0248

-   1. The DNA and protein sequence of Cj0588 are provided in SEQ ID    NO:3 and SEQ ID NO:4, respectively.-   2. The DNA and protein sequence of Cj0248 are provided in SEQ ID    NO:5 and SEQ ID NO:6, respectively.-   3. Mutation of C. jejuni genes

Genes Cj0588 and Cj0248 were cloned and mutated in C. jejuni byinsertion of a chloramphenicol cassette in the center of each gene.Essentially, two sets of primers were designed for each gene to yieldfragments consisting of the initial and terminal 15 bases of the geneand their associated flanking regions. These fragments were cloned intoa suicide vector and positioned around the chloramphenicolacetyltransferase (CAT) gene, such that the antibiotic cassette wasbetween the initial and terminal regions of the gene, taking up ˜255bases of each gene. The vector containing the CAT gene and the flankingbases of each gene was then introduced by electroporation into C. jejunistrain M129 (Cj0588) or strain NCTC11168 (Cj0248), and the mutationtransferred into the genome via a double crossover. Followingelectroporation the mutant strain was plated on Mueller Hinton agarcontaining chloramphenicol. Colonies that grew on the selective mediawere then confirmed by PCR to contain the cassette in the chromosome inthe proper direction. Colonies were then harvested and stored at −78° C.

-   4. Effects of the Cj0588 mutation on broiler colonization.

The ΔCj0588 mutant and M129 C. jejuni parent strain were each examinedfor colonization traits in poultry. Four separate studies wereconducted. The detection limit is less than 10 organisms. There was asignificant reduction in the colonization of birds inoculated with theM129::tlyA mutant, demonstrating that the C. jejuni Cj0588 gene isimportant in the colonization of poultry.

Study 1:

Chickens were obtained and housed as above. At 12 days, chickens wereorally inoculated with 1.0 ml of approximately 1.0×10⁵ per ml of viableC. jejuni M129 and the M129::tlyA mutant. At day 22 the birds werenecropsied, and Campylobacter was enumerated as above. There was asignificant reduction in the colonization of birds inoculated with theM129::tlyA mutant.

TABLE 4 Colonization of chickens with a C. jejuni parent M129 andM129::tlyA strain. M129::tlyA M129::tlyA M129 M129 52 ND 73 1.00E+02 53ND 74 ND 54 ND 75 2.00E+02 55 ND 76 ND 56 ND 77 4.20E+03 57 ND 78 ND 58ND 79 ND 59 ND 80 ND 60 ND 81 1.20E+04 61 ND 82 1.00E+04 TOTAL NDAverage 5.30E+03Study 2:

Chickens were obtained and housed as above. At 14 days, birds wereorally inoculated with 1.0 ml of approximately 1.0×10⁶ per ml of viableC. jejuni M129 and the M129::tlyA mutant. At day 28, the birds werenecropsied, and Campylobacter was enumerated as above. There was asignificant reduction in the colonization of birds inoculated with theM129::tlyA mutant.

TABLE 5 Colonization of chickens with a C. jejuni parent M129 andM129::tlyA strain. M129::tlyA M129::tlyA M129 M129 1 ND 1 2.30E+05 2 ND2 1.40E+05 3 ND 3 1.50E+03 4 ND 4 ND 5 ND 5 ND 6 ND 6 1.30E+06 7 ND 7 ND8 ND 8 1.90E+03 9 ND 9 1.80E+03 10  ND 10 ND 11  ND 11 8.70E+03 12  ND12 ND 13  ND 13 1.70E+03 14  ND 14 1.70E+03 15  ND 15 ND 16  ND 163.60E+03 17  ND 17 3.10E+03 TOTAL ND Average 1.54E+05Study 3:

Chickens were obtained and housed as above. At 14 days, birds wereorally inoculated with 1.0 ml of approximately 1.0×10⁸ per ml of viableC. jejuni M129 and the M129::tlyA mutant. At day 28, the birds werenecropsied, and Campylobacter was enumerated as above. There was asignificant reduction in the colonization of birds inoculated with theM129::tlyA mutant.

TABLE 6 Colonization of chickens with a C. jejuni parent M129 andM129::tlyA strain. M129::tlyA M129::tlyA M129 M129 1 ND 1 9.40E+04 2 ND2 2.10E+03 3 ND 3 2.10E+03 4 ND 4 1.00E+02 5 ND 5 4.70E+03 6 ND 62.60E+07 7 ND 7 3.70E+03 8 ND 8 1.10E+03 9 ND 9 4.00E+03 10  ND 102.70E+03 11  ND 11 2.80E+03 12  ND 12 1.50E+04 13  ND 13 2.10E+03 14  ND14 1.10E+03 15  ND 15 1.50E+05 16  ND 16 5.10E+03 17 2.00E+02 TOTAL NDAverage 1.55E+06Study 4

Chickens were obtained and housed as above. At 14 days, birds wereorally inoculated with 1.0 ml of approximately 1.0×10⁸ per ml of viableC. jejuni M129 and the M129::tlyA mutant. At day 28, the birds werenecropsied, and Campylobacter was enumerated as above. There was asignificant reduction in the colonization of birds inoculated with theM129::tlyA mutant.

TABLE 7 Colonization of chickens with a C. jejuni parent M129 andM129::tlyA strain. M129::tlyA M129::tlyA M129 M129  1 ND 1 1.10E+03  2ND 2 2.00E+04  3 ND 3 ND  4 ND 4 1.00E+02  5 ND 5 4.30E+03  6 ND 62.30E+03  7 ND 7 1.00E+02  8 ND 8 3.00E+02  9 ND 9 1.00E+02 10 ND 10 ND11 ND 11 2.30E+03 12 ND 12 2.80E+03 13 ND 13 1.70E+06 14 ND 14 1.10E+0315 ND 15 1.50E+03 16 ND 16 2.80E+03 17 ND 17 1.10E+04 18 ND 18 1.10E+0319 ND 19 7.00E+02 20 ND 20 2.50E+05 21 ND 21 1.10E+06 22 ND 22 ND 23 ND23 4.80E+03 TOTAL ND Average 1.35E+05

-   5. Effects of the ΔCj0248 mutation on broiler colonization.

The ΔCj0248 mutant and NCTC11168 C. jejuni parent strain were examinedfor colonization traits in poultry. Chickens were obtained and housed asabove. At day 14, the birds were orally inoculated with 0.5 ml ofapproximately 1.0×10⁵ per ml of viable C. jejuni strain NCTC11168(n=31), the ΔCj0248 mutant strain (n=30), or served as negativecontrols. At day 24, the birds were necropsied, and Campylobacter wasenumerated as above. There was a significant reduction in thecolonization of birds inoculated with the ΔCj0248 mutant strain. All thebirds inoculated with the wild-type strain NCTC11168 were colonized atan average of 7.42E+07 CFU/g, whereas, 3 of 30 birds (<10 CFUs) werecolonized at an average of 7.33E+01 CFU/g with the C. jejuni mutantstrain ΔCj0248 (Table 8).

TABLE 8 Colonization of Broilers with C. jejuni NCTC11168 Wild-type andMutant NCTC11168 Wild Type NCTC11168 Mutant Cj0248 Controls Group 1Group 2 Group 3 Group 1 Group 2 Group 3 ND 3.30E+07 2.30E+08 4.60E+05 NDND 2.00E+02 ND 4.10E+08 3.20E+07 8.00E+03 ND ND ND ND 1.60E+07 4.20E+051.00E+04 ND 1.90E+03 ND ND 2.40E+07 7.10E+07 1.20E+04 ND ND ND ND9.30E+05 8.20E+04 3.40E+07 ND ND 1.00E+02 ND 2.00E+06 9.50E+07 3.30E+05ND ND ND ND 1.40E+07 5.00E+06 3.10E+07 ND ND ND ND 9.50E+08 5.30E+071.50E+06 ND ND ND ND 5.90E+07 8.30E+07 1.00E+02 ND ND ND ND 2.40E+072.30E+04 3.10E+03 ND ND 1.30E+08 ND Average 1.51E+08 5.70E+07 6.73E+06ND 1.90E+02 3.33E+01 Average: Wild type = 7.42E+07; Mutant = 7.33E+01;ND = not detected, <10 CFU

-   6. Salmonella Typhimurium vector χ9992:

The vector used is a Salmonella Typhimurium vector χ9992, as describedabove.

-   7. Cloning and expressing C. jejuni genes into the Salmonella    vector.

Both genes Cj0588 and Cj0248 were cloned into the vector plasmid pYA3493and expressed from the attenuated Salmonella Typhimurium vector χ9992,as described above.

-   8. Vaccination of chickens with the attenuated Salmonella vector    expressing Cj0588 from plasmid pYA3493.

Chickens were obtained, housed, and vaccinated as above. Trial 1chickens were orally challenged with 1.0 ml of the homologous C. jejunistrain M129 (˜1×10⁷ CFU/ml) and trial 2 chickens were orally challengedwith 1.0 ml of a heterologous strain C. jejuni NCTC11168 (˜1×10⁵ CFU/ml)at 10 days after the final vaccination. Ten days post challenge, thechickens were necropsied, and Campylobacter enumerated as above.

In trial 1, a 4-log reduction of C. jejuni in cecal contents of chickensvaccinated with the vector expressing Cj0588 protein following challengewith the homologous M129 strain was observed, as compared to chickensreceiving the empty vector (EV) vaccine. A 2-log reduction in C. jejuniwas observed when the Cj0588 vaccinates were compared to the normalcontrols. In trial 2, an overall 1-log reduction of C. jejuni in cecalcontents of chickens vaccinated with the Cj0588 protein was observedfollowing challenge with a heterologous strain NCTC11168, as compared tochickens receiving the EV vaccine. These trials demonstrated asignificant reduction of C. jejuni in cecal contents of chickensvaccinated with the vector expressing the Cj0588 protein and challengedwith the homologous strain. In addition, a 1-log reduction was seen inchickens challenged with a heterologous strain. A greater reductionwould have occurred in trial 2 if not for three outliers (underlinedboxes) (Table 9).

TABLE 9 Cecal numbers of C. jejuni in vaccinated (Cj0588) and controlchickens C. jejuni M129 Challenge - Trial 1 C. jejuni NCTC11168Challenge - Trial 2 Control- Control- Cj0588 EV Controls Cj0588 Cj0588Cj0588 EV 6.00E+03 1.00E+04 2.00E+04 1.10E+03 5.00E+03 1.20E+05 5.00E+077.00E+02 2.00E+04 7.00E+04 1.50E+03 9.00E+04 1.00E+00 3.00E+07 7.00E+031.00E+04 1.00E+05 1.00E+02 3.00E+03 1.20E+04 2.00E+07 3.30E+03 1.00E+053.00E+04 1.00E+02 1.00E+00 6.00E+04 1.70E+08 2.00E+02 3.00E+04 3.00E+045.00E+03 4.00E+01 1.00E+02 3.00E+07 7.00E+02 3.00E+04 7.00E+04 1.00E+033.00E+07 4.00E+07 8.00E+03 1.00E+04 1.00E+04 2.00E+03 5.00E+01 2.70E+043.00E+03 3.00E+04 1.00E+06 7.00E+02 1.70E+04 6.00E+02 3.00E+03 3.00E+041.00E+04 2.00E+01 5.00E+07 7.00E+04 3.20E+03 5.00E+04 1.00E+04 5.00E+042.00E+02 1.20E+05 1.00E+03 2.30E+04 3.50E+05 7.00E+01 1.00E+00 2.00E+044.00E+03 1.00E+04 7.00E+04 5.60E+03 1.00E+00 2.00E+04 6.80E+03 1.00E+042.10E+06 Average 5.60E+03 6.68E+06 3.37E+06 6.00E+07 2.30E+03 3.00E+087.00E+04 Geomean 7.73E+02 1.24E+03 1.28E+04 4.33E+07 1.40E+03 1.00E+043.00E+05 7.00E+03 6.00E+05 Combined Trial 2 data Average 3.60E+032.00E+07 3.03E+05 Average 3.35E+06 6.00E+07 Geomean 2.47E+03 3.83E+048.36E+04 Geomean 2.31E+03 4.33E+07

Example 3 Dual Vaccination Using Cj0998c and Cj0588

Chickens were obtained, housed, and vaccinated as above, with theexception that each chicken received 2 ml of vaccine, 1 ml per antigen,at each vaccination. Chickens were orally challenged with 1.0 ml of thehomologous C. jejuni strain NCTC11168 (˜1×10⁵ CFU/ml) at 10 days afterthe final vaccination. Ten days post challenge the chickens werenecropsied, and Campylobacter enumerated as above. One vaccinate groupshowed a near total ˜7 log reduction in Campylobacter colonization,while colonization was reduced by ˜1 log in the other group, as comparedto empty vector and positive control groups.

Example 4 Water Vaccination Using Cj0998c

Chickens were obtained and housed as above, with the exception that nofasting period was observed prior to vaccination and that water wasremoved for two hours prior to vaccination. On days 10 and 16, ˜1×10¹⁰cfu of Salmonella expressing Cj0998c (prepared as above) was added to ˜2L of water in commercially available fountain waterers. Water wasreturned when all vaccine had been consumed (8-12 hours). Controlsreceived ˜1×10{circumflex over ( )}10 cfu empty vector Salmonella or novaccine orally. Chickens were orally challenged with 1.0 ml of thehomologous C. jejuni strain NCTC11168 (˜1×10⁵ CFU/ml) at 10 days afterthe final vaccination. At day 36, the chickens were necropsied andCampylobacter enumerated as above. Campylobacter colonization wasreduced in vaccinate groups by approximately 2.5 logs as compared toempty vector and positive control groups.

REFERENCES

-   Kong, Q., Six, D. A., Roland, K. L., Liu, Q., Gu, L., Reynolds, C.    M., Wang, X., Raetz, C. R., Curtiss, R., 3rd, 2011. Salmonella    synthesizing 1-dephosphorylated [corrected] lipopolysaccharide    exhibits low endotoxic activity while retaining its immunogenicity.    J Immunol 187, 412-423.-   Wyszynska, A., Raczko, A., Lis, M., Jagusztyn-Krynicka, E. K., 2004.    Oral immunization of chickens with avirulent Salmonella vaccine    strain carrying C. jejuni 72Dz/92 cjaA gene elicits specific humoral    immune response associated with protection against challenge with    wild-type Campylobacter. Vaccine 22, 1379-1389.-   Scallan E, Hoekstra R M, Angulo F J, Tauxe R V, Widdowson M A, Roy S    L, et al. Foodborne illness acquired in the United States—major    pathogens. Emerging infectious diseases. 2011; 17(1):7-15. Epub Jan.    5, 2011. doi: 10.3201/eid/1701.091101p1. PubMed PMID: 21192848.-   Buzby J C, Allos B M, Roberts T. The economic burden of    Campylobacter-associated Guillain-Barre syndrome. Journal of    Infectious Diseases. 1997; 176:S192-S7. PubMed PMID:    ISI:A1997YH50600023.-   Pope J E, Krizova A, Garg A X, Thiessen-Philbrook H, Ouimet J M.    Campylobacter reactive arthritis: a systematic review. Semin    Arthritis Rheum. 2007; 37(1):48-55. Epub Mar. 16, 2007. doi:    S0049-0172(07)00005-4 [pii]-   Li, Y., Wang, S., Scarpellini, G., Gunn, B., Xin, W., Wanda, S. Y.,    Roland, K. L., Curtiss, R., 3rd, 2009, Evaluation of new generation    Salmonella enterica serovar Typhimurium vaccines with regulated    delayed attenuation to induce immune responses against PspA.    Proceedings of the National Academy of Sciences of the United States    of America 106, 593-598.-   Curtiss III, R., S-Y. Wanda, B. M. Gunn, X. Zhang, S. A. Tinge, V.    Ananthnarayan, H. Mo, S. Wang, and W. Kong. 2009. Salmonella strains    with regulated delayed attenuation in vivo. Infect. Immun.    77:1071-1082.

We claim:
 1. An immunogenic composition, comprising (a) one or morepolynucleotide encoding two or more proteins including (i) a proteincomprising an amino acid sequence at least 80 percent identical to SEQID NO:2 (Cj0998c protein), or antigenic portions thereof, and (ii) oneor more proteins comprising an amino acid sequence at least 80 percentidentical to a protein selected from the group consisting of SEQ ID NO:6(Cj0248 protein), SEQ ID NO:142 (Cj1534c protein), SEQ ID NO:108(Cj1656c protein), SEQ ID NO:110 (Cj0428 protein), SEQ ID NO:112(Cj0168c protein), SEQ ID NO:114 (Cj0427 protein), SEQ ID NO:116 (Cj0113protein), SEQ ID NO:118 (Cj0982c protein), SEQ ID NO:120 (Cj0921cprotein), SEQ ID NO:122 (Cj1259 protein), SEQ ID NO:124 (Cj1339cprotein), SEQ ID NO:126 (Cj0034c protein), SEQ ID NO:128 (Cj0404protein), SEQ ID NO:130 (Cj0365c protein), and SEQ ID NO:132 (Cj0755protein), or antigenic portions thereof; and (b) a promoter operativelylinked to each of the one or more polynucleotide, wherein the promoterregion is capable of directing expression of the encoded protein(s). 2.The immunogenic composition of claim 1, wherein the compositioncomprises one or more polynucleotide encoding three or more of therecited proteins, or antigenic portions thereof.
 3. The immunogeniccomposition of claim 1, wherein the composition further comprises apolynucleotide encoding a protein comprising an amino acid sequence atleast 80 percent identical to SEQ ID NO:116 (Cj0113 protein), or anantigenic portion thereof.
 4. The immunogenic composition of claim 1,wherein the one or more polynucleotide encodes two or more proteinsincluding (i) a protein comprising the amino acid sequence of SEQ IDNO:2 (Cj0998c protein), or antigenic portions thereof, and (ii) one ormore proteins comprising the amino acid sequence of a protein electedfrom the group consisting of SEQ ID NO:4 (Cj0588 protein), SEQ ID NO:6(Cj0248 protein), SEQ ID NO:142 (Cj1534c protein), SEQ ID NO:108(Cj1656c protein), SEQ ID NO:110 (Cj0428 protein), SEQ ID NO:112(Cj0168c protein), SEQ ID NO:114 (Cj0427 protein), SEQ ID NO:116 (Cj0113protein), SEQ ID NO:118 (Cj0982c protein), SEQ ID NO:120 (Cj0921cprotein), SEQ ID NO:122 (Cj1259 protein), SEQ ID NO:124 (Cj1339cprotein), SEQ ID NO:126 (Cj0034c protein), SEQ ID NO:128 (Cj0404protein), SEQ ID NO:130 (Cj0365c protein), and SEQ ID NO:132 (Cj0755protein), or antigenic portions thereof.
 5. The immunogenic compositionof claim 1, wherein the immunogenic composition is present in anavirulent, non-Campylobacter bacterial carrier cell.
 6. The immunogeniccomposition of claim 5, wherein the avirulent, non-Campylobacterbacterial carrier cell is selected from the group consisting ofattenuated L. monocytogenes, attenuated Salmonella, attenuated V.cholerae, attenuated Shigella spp., attenuated M. bovis BCG, attenuatedY. enterocolitica, attenuated B. anthracis, S. gordonii, Lactobacillusspp., and Staphylococcus spp.
 7. The immunogenic composition of claim 5,wherein the avirulent, non-Campylobacter bacterial carrier cell is anattenuated Salmonella.
 8. The immunogenic composition of claim 4,wherein the immunogenic composition is present in an avirulent,non-Campylobacter bacterial carrier cell.
 9. The immunogenic compositionof claim 8, wherein the avirulent, non-Campylobacter bacterial carriercell is selected from the group consisting of attenuated L.monocytogenes, attenuated Salmonella, attenuated V. cholerae, attenuatedShigella spp., attenuated M. bovis BCG, attenuated Y. enterocolitica,attenuated B. anthracis, S. gordonii, Lactobacillus spp., andStaphylococcus spp.
 10. The immunogenic composition of claim 8, whereinthe avirulent, non-Campylobacter bacterial carrier cell is an attenuatedSalmonella.
 11. The immunogenic composition of claim 1, wherein theimmunogenic composition is formulated for oral delivery or mucosaldelivery.
 12. The immunogenic composition of claim 1, further comprisingan adjuvant.
 13. The immunogenic composition of claim 4, wherein theimmunogenic composition is formulated for oral delivery or mucosaldelivery.
 14. The immunogenic composition of claim 4, further comprisingan adjuvant.